• The Korean Journal of Physiology and Pharmacology
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• v.22 no.6
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• pp.617-625
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• 2018

Neddylation is a post-translational protein modification process. MLN4924 is a newly discovered pharmaceutical neddylation inhibitor that suppresses cancer growth with several cancer types. In our study, we first investigated the effect of MLN4924 on colon cancer cells (HCT116 and HT29). MLN4924 significantly inhibited the neddylation of cullin-1 and colon cancer cell growth in a time and dose-dependent manner. MLN4924 induced G2/M cell cycle arrest and apoptosis in HCT116 and HT29 cells. Moreover, MLN4924 also triggered autophagy in HCT116 and HT29 cells via suppressing the PI3K/AKT/mTOR pathway. Inhibiting autophagy by autophagy inhibitor 3-MA or ATG5 knockdown reversed the function of MLN4924 in suppressing colon cancer cell growth and cell death. Interestingly, MLN4924 suppresses colon cell growth in a xenograft model. Together, our finding revealed that blocking neddylation is an attractive colon cancer therapy strategy, and autophagy might act as a novel anti-cancer mechanism for the treatment of colon cancer by MLN4924.

• Biomolecules & Therapeutics
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• v.30 no.5
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• pp.465-472
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• 2022

Melanoma is one of the most aggressive skin cancers. Hypoxia contributes to the aggressiveness of melanoma by promoting cancer growth and metastasis. Upregulation of cyclin D1 can promote uncontrolled cell proliferation in melanoma, whereas stimulation of cytotoxic T cell activity can inhibit it. Epithelial mesenchymal transition (EMT) plays a critical role in melanoma metastasis. Hypoxia-inducible factor-1α (HIF-1α) is a main transcriptional mediator that regulates many genes related to hypoxia. CoCl₂ is one of the most commonly used hypoxia-mimetic chemicals in cell culture. In this study, inhibitory effects of IDF-11774, an inhibitor of HIF-1α, on melanoma growth and metastasis were examined using cultured B16F10 mouse melanoma cells and nude mice transplanted with B16F10 melanoma cells in the presence or absence of CoCl₂-induced hypoxia. IDF-11774 reduced HIF-1α upregulation and cell survival, but increased cytotoxicity of cultured melanoma cells under CoCl₂-induced hypoxia. IDF-11774 also reduced tumor size and local invasion of B16F10 melanoma in nude mice along with HIF-1α downregulation. Expression levels of cyclin D1 in melanoma were increased by CoCl₂ but decreased by IDF-11774. Apoptosis of melanoma cells and infiltration of cytotoxic T cells were increased in melanoma after treatment with IDF-11774. EMT was stimulated by CoCl₂, but restored by IDF11774. Overall, IDF-11774 inhibited the growth and metastasis of B16F10 melanoma via HIF-1α downregulation. The growth of B16F10 melanoma was inhibited by cyclin D1 downregulation and cytotoxic T cell stimulation. Metastasis of B16F10 melanoma was inhibited by EMT suppression.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.5
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• pp.401-408
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• 2009

$K^+$-$Cl^-$-cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase $A_2$ ($PLA_2$)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced $K^+$ efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7 -dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1Hinden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activatorinduced $K^+$ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calciumindependent $PLA_2$ ($iPLA_2$), whereas it was not significantly altered by arachidonyl trifluoromethylketone ($AACOCF_3$) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic $PLA_2$ ($cPLA_2$) and the secretory $PLA_2$ ($sPLA_2$), respectively. NEM increased AA liberation in a doseand time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas $AACOCF_3$ and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that $iPLA_2$-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.

• Journal of Life Science
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• v.21 no.5
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• pp.647-655
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• 2011

The neurotoxicity of aggregated amyloid ${\beta}$ ($A{\beta}$) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). It can cause neurotoxicity in AD by evoking a cascade of apoptosis to neuron. Here, we investigated the neuroprotective effects of cilostazol, which acts as a phosphodiesterase III inhibitor, on $A{\beta}_{25-35}$-induced cytotoxicity in mouse neuronal cells and cognitive decline in the C57BL/6J AD mouse model via peroxisome proliferator-activated receptor (PPAR)-${\gamma}$ activation. $A{\beta}_{25-35}$ significantly reduced cell viability and increased the number of apoptotic-like cells. Cilostazol treatment recovered cells from $A{\beta}$-induced cell death as well as rosiglitazone, a PPAR-${\gamma}$ activator. These effects were suppressed by GW9662, an antagonist of PPAR-${\gamma}$ activity, indicative of a PPAR-${\gamma}$-mediated signaling. In addition, cilostazol and rosiglitazone also restored PPAR-${\gamma}$ activity levels that had been altered as a result of $A{\beta}_{25-35}$ treatment, which were antagonized by GW9662. Furthermore, cilostazol also markedly decreased the number of apoptotic-like cells and decreased the Bax/Bcl-2 ratio. Intracerebroventricular injection of $A{\beta}_{25-35}$ in C57BL/6J mice resulted in impaired cognitive function. Oral administration of cilostazol (20 mg/kg) for 2 weeks before $A{\beta}_{25-35}$ injection and once a day for 4 weeks post-surgery almost completely prevented the $A{\beta}_{25-35}$-induced cognitive deficits, as did rosiglitazone. Taken together, our findings suggest that cilostazol could attenuate $A{\beta}_{25-35}$-induced neuronal cell injury and apoptosis as well as promote the survival of neuronal cells, subsequently improving cognitive decline in AD, partly because of PPAR-${\gamma}$ activation. The phosphodiesterase III inhibitor cilostazol may be the basis of a novel strategy for the therapy of AD.

• Biomolecules & Therapeutics
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• v.30 no.4
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• pp.348-359
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• 2022

Gastric adenocarcinoma is among the top causes of cancer-related death and is one of the most commonly diagnosed carcinomas worldwide. Benzyl isothiocyanate (BITC) has been reported to inhibit the gastric cancer metastasis. In our previous study, BITC induced apoptosis in AGS cells. The purpose of the present study was to investigate the effect of BITC on autophagy mechanism in AGS cells. First, the AGS cells were treated with 5, 10, or 15 μM BITC for 24 h, followed by an analysis of the autophagy mechanism. The expression level of autophagy proteins involved in different steps of autophagy, such as LC3B, p62/SQSTM1, Atg5-Atg12, Beclin1, p-mTOR/mTOR ratio, and class III PI3K was measured in the BITC-treated cells. Lysosomal function was investigated using cathepsin activity and Bafilomycin A1, an autophagy degradation stage inhibitor. Methods including qPCR, western blotting, and immunocytochemistry were employed to detect the protein expression levels. Acridine orange staining and omnicathepsin assay were conducted to analyze the lysosomal function. siRNA transfection was performed to knock down the LC3B gene. BITC reduced the level of autophagy protein such as Beclin 1, class III PI3K, and Atg5-Atg12. BITC also induced lysosomal dysfunction which was shown as reducing cathepsin activity, protein level of cathepsin, and enlargement of acidic vesicle. Overall, the results showed that the BITC-induced AGS cell death mechanism also comprises the inhibition of the cytoprotective autophagy at both initiation and degradation steps.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4331-4338
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• 2014

$N^1$-(2, 5-dimethoxyphenyl)-$N^8$-hydroxyoctanediamide (N25) is a novel SAHA cap derivative of HDACi, with a patent (No. CN 103159646). This invention is a hydroxamic acid compound with a structural formula of $RNHCO(CH_2)6CONHOH$ (wherein R=2, 5dimethoxyaniline), a pharmaceutically acceptable salt which is soluble. In the present study, we investigated the effects of N25 with regard to drug distribution and molecular docking, and anti-proliferation, apoptosis, cell cycling, and $LD_{50}$. First, we designed a molecular approach for modeling selected SAHA derivatives based on available structural information regarding human HDAC8 in complex with SAHA (PDB code 1T69). N25 was found to be stabilized by direct interaction with the HDAC8. Anti-proliferative activity was observed in human glioma U251, U87, T98G cells and human lung cancer H460, A549, H1299 cells at moderate concentrations ($0.5-30{\mu}M$). Compared with SAHA, N25 displayed an increased antitumor activity in U251 and H460 cells. We further analyzed cell death mechanisms activated by N25 in U251 and H460 cells. N25 significantly increased acetylation of Histone 3 and inhibited HDAC4. On RT-PCR analysis, N25 increased the mRNA levels of p21, however, decreased the levels of p53. These resulted in promotion of apoptosis, inducing G0/G1 arrest in U251 cells and G2/M arrest in H460 cells in a time-dependent and dose-dependent manner. In addition, N25 was able to distribute to brain tissue through the blood-brain barrier of mice ($LD_{50}$: 240.840mg/kg). In conclusion, our findings demonstrate that N25 will provide an invaluable tool to investigate the molecular mechanism with potential chemotherapeutic value in several malignancies, especially human glioma.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.287-294
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• 2011

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs, we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at $39^{\circ}C$ 5% $CO_2$ in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control ($6.0{\pm}1.0$ vs $3.3{\pm}0.6$, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ER-stress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

• Journal of The Korean Dental Society of Anesthesiology
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• v.14 no.3
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• pp.157-165
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• 2014

Background: Incisional site of surgical operation become transient ischemic state and then occur reoxygenation due to vasodilatation by inflammatory reaction, the productive reactive oxygen species (ROS) give rise to many physiologic results. Apoptosis have major role on elimination of inflammatory cell and formation of granulation tissue in normal wound healing process. Remifentanil can prevent the inflammatory response and can suppress inducible nitric oxide synthase expression in a septic mouse model. After cardiopulmonary bypass for coronary artery surgery, remifentanil can also inhibit the release of biomarkers of myocardial damage. Here we investigated whether remifentanil pretreatment has cellular protective effect against hypoxia-reoxygenation in HaCaT human keratinocytes, if so, the role of apoptosis and autophagy on this phenomenon. Methods: The HaCaT human keratinocytes were exposed to various concentrations of remifentanil (0.01, 0.05, 0.1, 0.5 and 1 ng/ml) for 2 h before hypoxia (RPC/HR group). These cells were cultured under 1% oxygen tension for 24h at $37^{\circ}C$. After hypoxia, to simulate reoxygenation and recovery, the cells were reoxygenated for 12 h at $37^{\circ}C$. 3-MA/RPC/HR group was treated 3-methyladenine (3-MA), autophagy inhibitor for 1h before remifentanil treatment. Cell viability was measured using a quantitative colorimetric assay with thiazolyl blue tetrazoliumbromide (MTT, amresco), showing the mitochondrial activity of living cells. To investigate whether the occurrence of autophagy and apoptosis, we used fluorescence microscopy and Western blot analysis. Results: The viability against hypoxia-reoxygenation injury in remifentanil preconditioning keratinocytes were increased, and these cells were showed stimulated expression of autophagy 3-MA suppressed the induction of autophagy effectively and the protective effects on apoptosis. Atg5, Beclin-1, LC3-II and p62 were elevated in RPC/HR group. But they were decreased when autophagy was suppressed by 3-MA. Conclusions: Remifentanil preconditioning showed the protective effect in human keratinocytes, and we concluded that autophagy may take the major role in the recovery of wound from hypoxia-reoxygenation injury. We suggest that further research is needed about the cell protective effects of autophagy.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.392-402
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• 2017

Background: Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. Methods: Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. $H_2O_2$ productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusion: Of the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-$\text\tiny L$-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.

• Journal of Oral Medicine and Pain
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• v.36 no.1
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• pp.11-20
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• 2011

Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent. And it is known that antitmor activity of VPA is associated with its targeted at histone deacetylases. 17AAG, Inhibition of HSP90 leads to the proteasome degradation of the HSP90 client proteins, such as Akt, Raf/Ras, Erk, VEGF, cyclin D and p53, and causes potent antitumor activity. It is reported that 17AAG-induced HSP90 inhibition results in prevention of cell proliferation and induction of apoptosis in several types of cancer. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and the HSP90 inhibitor, 17AAG on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and 17AAG showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-3, caspase-7 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or 0.5 ${\mu}M$ 17AAG for 48 h did not induce apoptosis, the co-treatment with them induced prominently apoptosis. Therefore our data in this study provide the possibility that combination therapy with VPA and 17AAG could be considered as a novel therapeutic strategy for human osteosarcoma.